The raf-1 oncogene is located at chromosome 3p25 and one allele of Raf-1 is deleted in small cell lung carcinoma and renal cell carcinoma. Raf-1 transcripts are expressed in all mouse tissues, but the levels between tissues vary and Raf-1 overexpression occurs in a variety of tumors. To identify factors regulating transcription from the Raf-1 promoter, we have functionally characterized the human Raf-1 promoter. The minimal raf-1 promoter is located within a 250-base pair (bp) fragment and contains one region that specifically bound nuclear proteins. This region, located between nucleotides +6 and +40, includes binding sites for E2F, HIP-1, and a transcriptional activation sequence found in the DHFR gene. An initiator (Inr)-like sequence is located at -3 which probably directs the start site of transcription. A second footprint, located at -150, contains palindromic Sp1 and Wilms' tumor (WT1) binding sites. Cotransfection of a wild-type WT1 expression vector with a Raf-1 promot- er/reporter construct decreased reporter expression sevenfold. Mutations in the WT1 binding site blocked binding of purified WT1 protein, and Raf-1 transcripts were overexpressed in sporadic Wilms' tumors. The 14 kb A-raf-1 gene is located at Xp11.2, where it resides in a gene cluster composed of Syn-1, TIMP, and A-raf near the translocation in synovial carcinoma. A-raf transcripts are predominantly expressed in urogenital tissues. The minimal functional A-raf promoter is located within a 120 bp fragment that interacts specifically with a nuclear factor(s). We have constructed a series of mini-gene plasmids which express either normal or mutationally altered forms of the Raf-1 cDNA under Raf-1 or A-raf promoter control. The Raf-1 driven constructs were more efficient than other expression vectors in transformation assays, suggesting a position independent effect. We have introduced these constructs into the mouse germ line.